Sam:LabNotes/Microbiome-new/2010-9-10: Difference between revisions

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>Sam Chiang
>Sam Chiang
 
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='''MDA amplicons shearing using Misonic sonicator 3000 - test2'''=
='''MDA amplicons shearing using Misonic sonicator 3000 - with 30sec(on/off) setting'''=


==Background==
==Background==
*The test on 09-09-'10 showed that amplicon was over-sheared using Palsson lab protocol (pulse on 1min, pulse off 10sec)
*The test on 09-09-'10 showed that amplicon was over-sheared using Palsson lab protocol (pulse on 1min, pulse off 10sec)
*I noticed that this sonificator is very similar to Bioruptor I used before. After checking Bioruptor manual, I found that it needs only <5min shearing for getting 1~2Kbfragments.
*I noticed that this sonificator is very similar to Bioruptor I used before. After checking Bioruptor manual, I found that it needs only <5min shearing for getting 1~2Kbfragments.
*An example of Bioruptor progarm for 300~400bp shearing.http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Human/2009-4-25
*'''An example of Bioruptor program for 300~400bp shearing.''' http://genome-tech.ucsd.edu/LabNotes/index.php/Sam:LabNotes/Human/2009-4-25
*'''An example of Bioruptor setting(Diagenode) '''[[File:DNA shearing using bioruptor.pdf]]


==Procedure==
==Procedure==
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**Preparing UV-ed ALS, NS buffer
**Preparing UV-ed ALS, NS buffer
   
   
  50uL-MDANo-SYBR recipe:
  50uL-MDA No-SYBR recipe:
                             1 rxn  4.5rxn         
                             1 rxn  4.5rxn         
  ---------------------------------------------
  ---------------------------------------------
Line 25: Line 26:
  Incubate at 30C for 8hr. Inactivate by 85C 3min.
  Incubate at 30C for 8hr. Inactivate by 85C 3min.


===Operation manual===
*Pool the four tubes of MDA amplicons into one and quantify the yield using QUBIT-HS dsDNA
*Hook up H20-tubes of cooling system. Release the white clipper.
**Result: 666ng/uL in 200uL.
*Fill up H2O(tap H2O is acceptable) in the cup-horn to match the yellow tags
*Take 1ug Amplicon and dilute into 100uL H2O in a 1.5mL-tube for shearing.
*Turn on the switch of the cooling system.
*Cooling system setting: Pump speed: "50", Desired temp "4C".
*Turn on the switch of main controller.  
*Program setting:
**Press "No" for not using micro-probe.
**Press "program" to set up new conditions.
**Tested condition: Pulse on(1min), Pulse(10sec), Output power(5), Temp monitoring(No). Total time(30min).
*Press "start" to start the shearing.
*Press "pause" to pause the shearing and aliqate sheared samples for validation.


*After using, use the opposite order of operation procedures to turn off the machine.
===Amplicon shearing test===
**Press "Stop" to stop shearing.
*Directly shear the raw MDA amplicons (1ug) without purification.
**Turn off the main controller.
*Following operation manual from 09/09/'10 with following changes on settings.
**Turn down pump speed to "0" and turn off the switch.
**Pulse on(30min), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(10min).
**For the cooling system, disconnect the H2O-tubes. Drain the H2O into the beaker.
**Take out 20uL (200ng) sample for validation every 2min.
**Wipe out spilled H2O.
 
*Technical support info: 800-323-4340 or 203-426-0101


==Results==
==Results==
*Sheared DNA were validated using 2% agarose recycled E-gel (20uL/well)
*Sheared DNA were validated using 2% agarose recycled E-gel (20uL/well)(200ng/well)
  [[File:sam091010-sheared DNA.jpg|400px]]
  [[File:sam091010-E gel.jpg|400px]]
 
*The 2min shearing with 30sec (on/off) setting can get the 1~2Kb fragment size, which is suitable for Sanger sequcncing.
*It seems that even 7min (in the current pulse condition) is still too much for getting a 1~2Kb size fragments.
**This result is reasonable because the Manual of Bioruptor sonicator(a very similar device)suggests to shear <5 min to get a 2~3Kb size (with 30sec on and off pulse).
*I will try to optimize the shearing setting using new samples.

Latest revision as of 18:37, 12 October 2010

MDA amplicons shearing using Misonic sonicator 3000 - with 30sec(on/off) setting[edit]

Background[edit]

Procedure[edit]

  • Prepare more E.coli gDNA MDA amplicons for testing.
    • Preparing UV-ed ALS, NS buffer
50uL-MDA No-SYBR recipe:
                            1 rxn   4.5rxn         
---------------------------------------------
RT-PCR grade H2O           29.0    130.5        
10x RepliPhi phi-29 buffer  5.0     22.5
1mM  N6 primer              2.5     11.25
RepliPhi Phi-29 (100U/ul)   2.5     11.25    
dNTP (25mM)                 2.0      9.0 
----------------------------------------------
                           41.0    184.5/4.5=41 

3uL ALS buffer + 3uL E.coli gDNA (1ng/uL) + 3uL NS buffer 
Transfer 41uL master mix into lysed template.
Incubate at 30C for 8hr. Inactivate by 85C 3min.
  • Pool the four tubes of MDA amplicons into one and quantify the yield using QUBIT-HS dsDNA
    • Result: 666ng/uL in 200uL.
  • Take 1ug Amplicon and dilute into 100uL H2O in a 1.5mL-tube for shearing.

Amplicon shearing test[edit]

  • Directly shear the raw MDA amplicons (1ug) without purification.
  • Following operation manual from 09/09/'10 with following changes on settings.
    • Pulse on(30min), Pulse off(30sec), Output power(5), Temp monitoring(No). Total time(10min).
    • Take out 20uL (200ng) sample for validation every 2min.

Results[edit]

  • Sheared DNA were validated using 2% agarose recycled E-gel (20uL/well)(200ng/well)
File:Sam091010-E gel.jpg
  • The 2min shearing with 30sec (on/off) setting can get the 1~2Kb fragment size, which is suitable for Sanger sequcncing.